Members of the CD1 family of MHC-like molecules specialize in presenting lipid antigens to T cells, in a process requiring endosomal trafficking, which is regulated by intrinsic tyrosine motifs encoded in their cytoplasmic tails. Recent studies in our laboratory have uncovered a new, important player in the mouse CD1d antigen presentation pathway, the invariant chain (Ii). Ii-deficient mice also present striking abnormalities affecting splenic marginal zone B cells (MZB), a population of CD1d high B cells that is recognized by CD1d autoreactive NKT cells. The general aim of our studies is to understand the molecular and cellular basis of CD1d-mediated lipid antigen presentation. The specific aims of this application will explore these newly uncovered functions of Ii in the CD1d presentation pathway. 1. Specific aim 1. Regulation of CD1d trafficking and antigen presentation in cell lines, defining (1.1) the dynamics of CD1d trafficking, and its association with Ii and other accessory molecules; (1.2) the contribution of Ii and the CD1d tail sorting motif to trafficking and antigen presentation; (1.3) the regulation of other CD1 isotypes by Ii (human CD Ia, b, c, d). 2. Specific aim 2. Regulation of CD1d trafficking and antigen presentation in mutant mice. Using mice that lack the tail sorting motif of CD1d (CD1-TD knock-in) and other antigen presentation mutant mice (Ii, H-2M, H-20), we will define their contribution to (2.1) CD1d trafficking in various fresh cell types; (2.2) CD1d mediated antigen presentation to T cells; (2.3) positive selection and maintenance of CD1 d-restricted T cells. 3. Specific aim 3. Regulation of CD1d high MZB cells in antigen presentation mutant mice. To understand how Ii regulates the development of CD1d high MZB cells, we will (3.1) study the development of MZB cells in Ii, CD1d, H-2M, H-20, IAcx, IABeta and CUTA deficient mice; (3.2) genetically dissect the Ii associated MZB phenotype by crossing Ii deficient mice to mice lacking Ii partners such as CD1d, H-2M and IA, and to BCR transgenic mice expressing MZB-included or excluded specificities.